Abnormalities in pancreatic beta-cell insulin release mechanisms can lead to serious metabolic disorders, such as diabetes mellitus. The secretion-stimulus coupling mechanism for nutrient induced insulin release is only partially understood. For glucose-stimulated insulin release, it has been revealed that glucose metabolism is necessary in order to evoke certain ion channel and electrical signaling activity at the beta-cell plasma membrane that then leads to a rapid rise in cytosolic [Ca++]i, which has long been presumed to be the signal to stimulate insulin release. However, it is unclear as to how a rise in intracellular [Ca++]i somehow acts to stimulate secretory granule movement towards the plasma membrane, fusion of the secretory granule and plasma membranes, and insulin exocytosis. There is a growing opinion that cytosolic [Ca++]i may not be a signal per se, but rather a simple permissive co-factor that provides the necessary intracellular Ca++-environment for Ca++-dependent exocytosis. It is clear that other yet to be defined factors, generated from beta-cell metabolism, are required to trigger insulin exocytosis. Furthermore, there is poor understanding of beta-cell exocytotic machinery, and how insulin exocytosis is triggered. A long-term objective of my research is to better define the molecular mechanism underlying insulin exocytosis, and how the 13-cell exocytotic machinery is regulated by a signal(s) derived from beta- cell nutrient metabolism. This broad objective is beyond the scope of a single proposal, so to gain a handle on beta-cell exocytosis we are currently focusing on the role of rab-3a, that is a prime protein candidate member of the exocytotic machinery in neuroendocrine cells. Our aims are to characterize the functional role of rab-3a in regulated beta-cell exocytosis, identify certain 'effector proteins' that specifically associate with rab-3a, and to gain an insight whereby the mechanism of rab-3a activating the beta-cell exocytotic machinery can be linked to metabolic signals that induce insulin exocytosis. Rab-3a protein is expressed in beta-cells and is mostly thought to be associated with insulin secretory granules. We have preliminary evidence which implicates a functional role of rab-3a in insulin exocytosis, in that synthetic peptides which mimic the effector domain of rab-3a directly stimulate insulin release in a Ca++-independent fashion from semi-permeabilized beta-cells. It is thought that the effector domain of rab-3a interacts with an unknown effector protein (a putative 'rab3a-receptor') to mediate triggering of exocytosis. By using novel photoaffinity cross-linking technology we have recently discovered such a putative 'rab-3a-receptor' as a 59kDa insulin secretory granule membrane protein that specifically associates with synthetic rab-3a effector domain peptides. We are currently trying to identify this 59kDa protein, and pursue further characterization of the beta-cell exocytotic machinery, using rab-3a as a 'handle'. This should provide a means whereby a link for beta- cell metabolic signaling effecting beta-cell exocytotic machinery can be better studied. It is intended that this will eventually lead to a more complete understanding of the secretion-stimulus coupling mechanism for insulin release.